Incorrect regulation of mitosis contributes to progression of cancer and birth defects and discoveries regarding the regulation of mitosis have positively impacted medical research and disease treatments. However, the potential for new insights and further impact on medicine clearly exists. For example, one of the most dramatic yet least understood mitotic processes is the disassembly and reassembly of nuclear pore complexes (NPCs), massive structures providing regulated gateways across the nuclear envelope. How this process is regulated and integrated with other mitotic events potentially involves reversible phosphorylation and our planned experiments will help test this hypothesis. We have shown the NIMA mitotic kinase of Aspergillus nidulans initiates mitosis by promoting NPC disassembly. NIMA is both required and sufficient to promote NPC disassembly and markedly affects mitosis in vertebrate cells suggesting the existence of conserved mitotic substrates. A. nidulans therefore provides a powerful and sophisticated model system in which to understand mitotic regulation of NPCs. The aims of this application include defining the dynamic mitotic changes in the NPC interactome using affinity purifications and mass spectrometry to identify new NPC proteins (Nups) as well as Nup associated proteins involved in mitotic regulation. In addition we will also focus studies on a new Nup associated protein (AN0162) which contains a transmembrane domain and potentially encodes a fourth transmembrane Nup. Our data suggest that AN0162 has a mitotic function to maintain the peripheral mRNA export factor Gle1 on the nuclear envelope surrounding the nucleolus during mitosis and we will test this hypothesis. Our third focus is on the mitotic functions of Nup2 and its associated proteins NupA, importin 1 and importin 2. Nup2 and NupA translocate from NPCs to chromatin during mitosis when they also become phosphorylated. We will test the hypothesis that NupA mediates the mitotic chromatin location of Nup2, which in turn controls the functions of importin 1 and 2 to regulate mitotic progression. As phosphorylation appears integral to this process, the final aim is to map mitotic phosphorylation sites within Nup2, NupA and importin 2 and then carry out in vivo mutational analysis of the specific phosphorylation sites to determine their function.